Physiol. Res. 60: 309-315, 2011
Monoamine Oxidase and Semicarbazide-Sensitive Amine Oxidase Kinetic Analysis in Mesenteric Arteries of Patients With Type 2 Diabetes S. F. NUNES1, I. V. FIGUEIREDO1, 3, J. S. PEREIRA2, E. T. DE LEMOS3, F. REIS3, F. TEIXEIRA3, M. M. CARAMONA1 1
Laboratory of Pharmacology, Faculty of Pharmacy, Coimbra University, Coimbra, Portugal, Portuguese Oncology Institute of Coimbra, Portugal, 3Institute of Pharmacology and Experimental Therapeutics, IBILI, Faculty of Medicine, Coimbra University, Coimbra, Portugal
2
Received February 22, 2010 Accepted August 30, 2010 On-line November 29, 2010
Summary
Corresponding author
Monoamine oxidase (MAO, type A and B) and semicarbazide-
M. M. Caramona, Laboratory of Pharmacology, Faculty of
sensitive amine oxidase (SSAO) metabolize biogenic amines,
Pharmacy, Coimbra University, 3000-548, Coimbra, Portugal.
however, the impact of these enzymes in arteries from patients
Fax: +351 239 488 503. E-mail:
[email protected]
with type 2 diabetes remains poorly understood. We investigated the kinetic parameters of the enzymes to establish putative correlations with noradrenaline (NA) content and patient age in
Introduction
human mesenteric arteries from type 2 diabetic patients. The kinetic parameters were evaluated by radiochemical assay and NA content by high-performance liquid chromatography (HPLC). The activity of MAO-A and SSAO in type 2 diabetic vascular tissues was significantly lower compared to the activity obtained in
non-diabetic
tissues.
In
the
correlation
between
MAO-A (Km) and NA content, we found a positive correlation for both the diabetic and non-diabetic group, but no correlation was established for patient age. In both groups, MAO-B (Vmax) showed a negative correlation with age. The results show that MAO-A and SSAO activities and NA content of type 2 diabetic tissues are lower compared to the non-diabetic tissues, while MAO-B activity remained unchanged. These remarks suggest that MAO-A and SSAO may play an important role in vascular tissue as well as in the vascular pathophysiology of type 2 diabetes. Key words Human
mesenteric
arteries
•
Monoamine
oxidase
•
Semicarbazide-sensitive amine oxidase • Type 2 diabetes • Noradrenaline content
Oxidative stress can be defined as a disturbance in the balance between the production of free radicals – such as superoxide anion, hydroxyl radicals and hydrogen peroxide – and antioxidant mechanisms (Ramakrishna and Jailkhani 2008). These pathways also include monoamine oxidases (A and B) and semicarbazidesensitive amine oxidase (SSAO) enzymes which take part in pathological changes, such as cardiac diseases and diabetic complications (Bianchi et al. 2005, Obata 2006). Monoamine oxidase (MAO, EC 1.4.3.4) catalyzes the oxidative deamination of biogenic amines (adrenaline, noradrenaline, serotonin, dopamine, tyramine and tryptamine) to the corresponding aldehyde, hydrogen peroxide (H2O2) and ammonia (Nagatsu 2004). In vitro, serotonin and noradrenaline (NA) are the preferential MAO-A substrates and the MAO-A enzyme is inhibited by clorgyline, whereas β-phenylethylamine is a substrate for MAO-B and selegiline as a selective MAO-B inhibitor. MAO (type A and B) functions include catabolism of exogenous amines, regulation of neurotransmitter levels and control of intracellular amine
PHYSIOLOGICAL RESEARCH • ISSN 0862-8408 (print) • ISSN 1802-9973 (online) © 2011 Institute of Physiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax +420 241 062 164, e-mail:
[email protected], www.biomed.cas.cz/physiolres
310
Nunes et al.
stores. Thus, it might be expected that their distribution in central nervous system and in other tissues is a reflection of these physiological roles (Billett 2004). The semicarbazide-sensitive amine oxidase enzyme (SSAO, EC 1.4.3.6) catalyzes the oxidative deamination of primary amines, such as methylamine or aminoacetone, to produce the corresponding aldehyde (formaldehyde and methylglyoxal, respectively) and converts circulating aliphatic and aromatic amines (tyramine, tryptamine, histamine and dopamine) into the corresponding aldehyde, H2O2 and ammonia, but is relatively inactive for NA and serotonin (Elliott et al. 1989, Obata 2006). The SSAO enzyme is present in blood, associated with cell membranes, such as endothelial and smooth muscle cells of blood vessels, and is selectively inhibited by semicarbazide (del Mar Hernandez et al. 2005). This enzyme is also known as vascular adhesion protein-1 (VAP-1) and plays an important role in the adhesion and migration of leukocytes to sites of inflammation (Bour et al. 2009). The mesenteric innervation system plays an important role in controlling mesenteric capacitance, and there are some studies reporting MAO (type A and B) and SSAO activity in non-diabetic patients (Figueiredo et al. 1998, Birch et al. 2008). However, the impact of those enzymes in the inferior mesenteric arteries of patients with type 2 diabetes remains poorly understood. The present study aims to determine (Km) and (Vmax) for the three enzymes, as well as to determine the NA content in inferior mesenteric arteries, in order to establish correlations between MAO-A and MAO-B with NA content, and also between MAO enzymes and patient age. A putative correlation can help to understand the influence of MAO and SSAO enzymes in the pathophysiological damage in the arterial walls from patients with type 2 diabetes.
Materials and Methods Study population Segments of human inferior mesenteric arteries from 12 non-diabetic patients (average age: 62.2±10.9 years old, 7 men and 5 women) and 8 patients with type 2 diabetes (average age: 63.1±6.2 years old, 5 men and 3 women), obtained from patients submitted for colorectal surgery, were provided by the Surgery Department of the Portuguese Oncology Institute of Coimbra. The study was approved by local medical ethics committee following the rules from Declaration of
Vol. 60 Helsinki of the World Medical Association, and all participants provided informed consent. Only nonsmokers participated in the study, and none of the participants suffered from thyroid dysfunction or liver disorders. The population included in this study consisted of patients with sigmoid or rectum carcinoma who were not subject to neo-adjuvant therapeutics and who presented surgery as the primary option. The type of surgical intervention used depended on the location of the tumor and whether sigmoidectomy or anterior recession of the rectum was carried out. The non-diabetic patients were selected according to the following criteria: blood glucose concentrations 6 % and on treatment with an oral antidiabetic drug. Vessels of greatest calibre (namely the inferior mesenteric artery) were selected as study material; in order to avoid interference from pathological anatomy. Only macroscopically healthy vessels were used. The arteries were placed in cold physiological saline solution and immediately transported to the laboratory where ±200 mg of each artery were homogenized 1:10 (w/v) with a concentric glass homogenizer in 10 mM sodium phosphate buffer (pH 7.4) at 4 ºC and the supernatant from the 600 g spin was taken and stored at −80 ºC until further analysis. Determination of MAO (A and B) and SSAO in the vascular tissues MAO (A and B) and SSAO activities were determined by radiochemical methods using 3H-5hydroxytryptamine creatinine sulphate (3H-5-HT), in a concentration range between 50-1 000 μM for MAO-A, 14 C-β-phenylethylamine hydrochloride (14C-β-PEA), 5-160 μM for MAO-B and 14C-benzylamine (14C-BZ), in a concentration range 50-1 600 μM for SSAO (Figueiredo et al. 1998). 5-HT, β-PEA and BZ were purchased from Sigma-Aldrich (Madrid, Spain), 3H-5-HT (15.1 Ci/mmol) and 14C-BZ (54 mCi/mmol) from Amersham International (London, United Kingdom) and 14 C-β-PEA (43.8 mCi/mmol) from Perkin Elmer LAS (Boston, MA). The MAO inhibitors and all other reagents used were of analytical grade. The tissue homogenates (25 μl) were preincubated for 20 min, at 37 ºC, with selegiline (10-4 M) as MAO-B inhibitor or clorgyline (10-6 M) as MAO-A
2011
MAO & SSAO Kinetic Analysis in Arteries of Diabetic Patients
311
Table 1. MAO-A, MAO-B and SSAO kinetic parameters in mesenteric arteries homogenates of non-diabetic group and patients with type 2 diabetes.
MAO-A MAO-B SSAO
Vmax (nmol.mg protein−1.h−1) Km (μM) Vmax (nmol.mg protein−1.h−1) Km (μM) Vmax (nmol.mg protein−1.h−1) Km (μM)
Non-diabetic group (n=12)
Type 2 diabetic group (n=8)
60.06 ± 21.43 178.30 ± 99.17 15.20 ± 3.03 100.50 ± 38.87 229.10 ± 46.28 301.00 ± 127.30
32.13 ± 18.24* 200.90 ± 122.60 12.31 ± 6.87 100.40 ± 59.68 148.30 ± 39.11* 228.10 ± 134.30
Values are means ± S.D. *P0.05) for both, non-diabetic and type 2 diabetic tissues, respectively. The MAO-A (Vmax) results in the mesenteric arteries from patients with type 2 diabetes (n=8) showed significant lower values compared to the non-diabetic tissues (n=12) (32.13±18.24 vs. 60.06±21.43 nmol.mg protein−1.h−1, P
